Foxp3+ T regulatory cells (Tregs) are critical for the maintenance of immune homeostasis. One of the major unresolved issues regarding their function is whether they can mediate antigen-specific suppression and what mechanism they utilize to perform this function. Several mechanisms have been proposed for the Treg-mediated suppression that can target both Teffector cell function and antigen presentation. These include: production of tolerogenic molecules i.e. IL-10, TGF-beta, IL-35; consumption of IL-2 via high affinity IL-2 receptors; CTLA-4 mediated inhibition of costimulation ; and contact-dependent killing of antigen presentation through Granzyme and perforin. All of these mechanisms are compatible with the paradigm of bystander suppression as suggested by the studies that Tregs primed by one antigen could subsequently suppress T cell proliferative responses to any other unrelated antigen activated in the same culture. However, these potential mechanisms for Treg suppression have been primarily derived from in vitro studies and it is clear that the mechanisms of in vivo regulation are likely to be much more complex. The goals of the present study were to first analyze the fine specificity of antigen specific Treg-mediated inhibition of priming naive T conventional (Tnaive) cells in vivo in mice and to compare the results of these studies with antigen-specific Treg-mediated suppression in vitro. We observed a dichotomy between the specificity of suppression in vitro and in vivo. In line with previous observations antigen-specific iTregs following activation by double-pulsed DC were capable of suppressing the expansion of Tnaive specific for their cognate antigen as well as Tnaive specific for an unrelated antigen in vitro. In contrast, when similar cell populations were transferred in vivo, iTregs activated by double-pulsed DC could only suppress Tnaive specific for their cognate antigen. To explore the mechanisms involved in Treg-mediated antigen-specific suppression in vivo, we performed an in-depth analysis of the physical interactions of antigen-specific Tregs with DC in vitro and in vivo to characterize potentially unique interactions of the Tregs with DCs. Using high resolution scanning electron microscopy (SEM) and transmission electron microscopy (TEM), we demonstrated that antigen specific iTregs acquire distinct morphology upon interaction with DCs displaying prominent uropods and synaptic filopodia, whereas in vitro activated antigen-specific T cells (Tactivated) and Tnaive preserved their round morphology and lacked the unique synaptic membrane features. Antigen-specific iTregs formed larger clusters around DCs and displayed wider membrane fusion sites compared to Tactivated or Tnaive. Using dynamic confocal imaging of co-cultures in vitro and intravital two photon microscopy of adoptively transferred antigen-pulsed DC and antigen-specific T cells, we demonstrated that antigen-specific iTregs displayed higher colocalization volume and longer durations of interaction than Tactivated or Tnaive. Lastly, we demonstrate that iTregs mediate a selective preclusion of Tnaive from their cognate antigen displayed on the DC surface, but allow bystander Tnaive cells to access the DC surface. While a selective exclusion of Tnaive that recognize the same antigen as the iTreg seems to be a plausible explanation for antigen-specific inhibition, the interaction of Treg with DC displayed further complexity. iTreg pretreatment of double pulsed DCs in vitro disabled the capacity of the DC to activate Tnaive specific for the antigen recognized by the iTreg, but not the response of Tnaive specific for an unrelated antigen expressed on the same DC surface. These studies suggested that Tregs use suppressor mechanisms in addition to preventing access of T effectors to antigen expressed on the DC surface. We demonstrate that antigen-specific iTregs could remove peptide-MHC Class II (pMHCII) complexes from the DC surface and thereby decrease the capacity of the DC to present antigen. Most importantly, the removal of pMHCII complexes was antigen-specific as capture of pMHCII complexes could only be detected when iTregs were specific for their target antigen and not for an unrelated antigen expressed on the same DC.